1. Field of the Invention
The present invention relates to a bacterium for producing polyhydroxyalkanoate in which a gene encoding acetyl-CoA acyltransferase is disrupted and a method for producing a polyhydroxyalkanoate using the above-mentioned bacterium for producing polyhydroxyalkanoate. In addition, the present invention relates to a gene targeting vector for disrupting a gene encoding acetyl-CoA acyltransferase of a bacterium for producing polyhydroxyalkanoate and a process for disrupting a gene encoding acetyl-CoA acyltransferase of a bacterium for producing polyhydroxyalkanoate using the above-mentioned gene targeting vector.
2. Related Background Art
There have been reported till now that many bacteria produce and accumulate poly-3-hydroxybutyric acid (PHB) or other poly-3-hydroxyalkanoates (PHA) in bacterial cells (“Handbook of Biodegradable Plastics”, ed. by Research Group on Biodegradable Plastics, NTS Co., Ltd., P178-197 (1995)). These polymers can be used for the production of various products through melt processing and the like similarly as conventional plastics. Furthermore, they have an advantage that they are completely decomposed in nature by bacteria because they are biodegradable, and they do not remain and cause pollution in natural environments like many conventional synthetic polymer compound. In addition, they are excellent in biocompatibility, and application as soft members for medical use is also expected. Particularly, considering wide application of PHA produced by microorganisms, for example, application as functional polymers, it is recently expected that “unusual PHA”, PHA in which a substituent group other than alkyl group is introduced into the side chain, is extremely useful. Examples of such a substituent group include those containing an aromatic ring (phenyl group, phenoxy group, benzoyl group, etc.) and unsaturated hydrocarbons, ester group, allyl group, cyano group, halogenated hydrocarbons, epoxides and thioethers. It is known that PHA produced by microorganisms can have various compositions and structures by the type of bacteria, medium composition, culture condition, etc. to be used for the production thereof, and various researches have been conducted on the bacteria which produce such PHA and biosynthetic pathway of PHA has been comparatively well investigated. So far, three pathways have been mainly proposed as the biosynthetic pathway of PHA by microorganisms.
The “first pathway” is a pathway synthesizing poly-3-hydroxybutyric acid (PHB), in which acetyl-CoA produced through glycolysis from sugar is condensed by β-ketothiolase to give acetoacetyl-CoA which is converted to (R)-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase and then converted to PHB by polyhydroxybutyrate synthetase.
The “second pathway” is one in which (R)-3-hydroxyacyl-ACP produced through fatty acid synthesis pathway from sugar is converted to (R)-3-hydroxyacyl-CoA by (R)-3-hydroxyacyl-ACP-CoA transferase, and this serves as a substrate of polyhydroxyalkanoate synthetic enzyme, and is converted to PHA (here, ACP is the abbreviation of acyl carrier protein.).
Whereas the starting material of the first and second pathways is a sugar, the “third pathway” is one in which trans-2,3-dehydroacyl-CoA, (S)-3-hydroxyacyl-CoA or 3-ketoacyl-CoA produced through β-oxidation pathway from fatty acid is converted to (R)-3-hydroxyacyl-CoA respectively by (R)-enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase or ketoacyl-CoA reductase, and this serve as a substrate of polyhydroxyalkanoate synthetase and is converted to PHA.
Generally, when an unusual PHA is to be produced by microorganisms, PHA producing bacteria are cultured with an alkanoate having a substituent group to be introduced added to the culture broth. Therefore, alkanoates having an unusual substituent group will be synthesized into PHA mainly through the “third pathway” using β-oxidation system. The outline of the third pathway is shown in FIG. 2.
In order to stably obtain PHA (in particular, unusual PHA) expected as a functional polymer in low cost and large quantities, it is necessary to achieve optimization as a whole of the flow of the biochemical transformation which intermediate metabolites constituting PHA synthesis/metabolic system in bacteria are subjected to so that PHA productivity may be increased. From this point of view, when an unusual PHA is produced by microorganisms, a method of using an alkanoate having a substituent group to be introduced as carbon source for replication as well as a raw material of polymer has been used for the purpose of improving the production.
Also commonly used is a method comprising extracting PHA after culturing a microorganism in a culture medium in which fatty acids of middle chain length such as octanoic acid and nonanoic acid are allowed to coexist as carbon source for replication in addition to the alkanoate having a substituent group to be introduced.
It is also shown in Journal of Bacteriology 182, 2978-2981 (2000) that intracellular PHA (usual mcl PHA) content (% cell dry weight) was able to be improved as a result of transforming fadA (acetyl-CoA acyltransferase) gene disrupted strain of “Escherichia coli bacterium” with PhaC2 (PHA synthetase derived from Pseudomonas oleovolans) and fabG (3-ketoacyl-CoA reductase derived from Pseudomonas aerugjnosa PAO1).
(Non-patent document 1) “Handbook of Biodegradable Plastics”, ed. by Research Group on Biodegradable Plastics, NTS Co., Ltd., P178-197 (1995).
(Non-patent document 2) Journal of Bacteriology 182,2978-2981(2000)